Avatar is developing HIV vaccine immunogens based on dityrosine (DT) crosslinking of soluble, native, Env gp140 trimers. Several of the most potent broadly neutralizing Abs (bnAbs) preferentially bind native Env trimers, and a trimeric immunogen thus would both present a wide range of key neutralizing epitopes, and simultaneously focus immune responses away from immunodominant, non-neutralizing epitopes. Developing a soluble trimeric immunogen, however, remains hampered by instability of the Env trimer complex. Avatar's DT-trimers are stabilized at the apex of the spike, thereby enabling full cleavage, while locking key, metastable neutralizing epitopes in their neutralizing conformation, thus focusing and optimizing the Ab response. In our Phase I studies, we introduced targeted DT crosslinks into gp140 trimers; and demonstrated that our DT-Env trimers retain native antigenicity. Our Phase II studies will further optimize the design of DT-Env trimer immunogens by transferring DT crosslinking into Envs from multiple Clade A, B and C strains and testing mutations that affect conformational heterogeneity. These studies and accompanying optimization of expression, purification, and crosslinking processes will enable the selection of 3-4 optimized candidate immunogen(s) for preclinical testing in Phase III. The project breaks into three Specific Aims: (i) immunogenicity testing of Phase I DT-Env trimers; (ii) immunogen optimzation, including strain and conformational optimization of the variable regions at the apex of the spike followed by further immunogenicity testing; and (iii) production optimization, including optimization of expression, purification, and the DT crosslinking reaction. Innovations: Avatar for the first time has rendered targeted DT crosslinking feasible, while retaining the structural and functional integrity of the gp140 Env trimer, and also improving thermostability. Targeted crosslinks are introduced after the protein/complex is fully folded at th apex of the trimer: immunogens are locked in antigenically favorable conformations, designed to optimize the display of key protective epitopes, while focusing the immune response away from immunodominant, non-neutralizing epitopes. Significance: the design of compact and stable DT-gp140 trimeric immunogens will focus immune responses toward broadly neutralizing - and away from non-neutralizing - epitopes. DT-mediated conformational locking of select Env conformers may further have the potential to enable 'single conformer' immunogens. The project will be executed in collaboration between Avatar Medical, LLC and the Pinter lab at PHRI/UMDNJ. The main goal is to select and produce 3-4 high-qualify candidate DT-Env gp140 trimeric immunogens during Phase II in preparation for preclinical development, and final down-selection of a final clinical candidate in Phase III.